| Dengue virus (DENV) is an enveloped virus belonging to mosquito-borne flaviviruses and possesses a positive stranded RNA genome. And DENV has 4 distinct serotypes (DENV1～4) derived from the different of envelop protein. DENV is transmitted by Aedes mosquitoes and Aedes aegypti, and it causes infections which leading to clinical symptoms ranging from self-limited, acute, febrile disease called dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Over 2.5 billion people, in more than 100 countries, are at risk of dengue infection, with millions of cases occurring around the world every year in the tropics and subtropics worldwide. Now WHO already put DHF/DSS, Hepatitis, Malaria and Tuberculosis into the list of the most four serious infectious diseases of the whole world. However, the pathogenesis of DENV infection is not clear, and there is not an efficient way to deal with the disease in the clinic therapy.Epidemiological studies indicated that DHF/DSS was more commonly seen during secondary infection with different serotype of DENV from that caused the primary infection. Antibody dependent enhancement (ADE) in DENV infection was suggested as one of explanation for this phenomenon and became a barrier for development of DENV vaccine. And there are two more mechanisms about the pathogenesis of DHF/DSS: (1) the virulence or replication ability of the virus; (2) immuno-pathogenesis theory: the cross reactive T-cell results in the immuno-damage. Recently the high titer of virus, secondary infection with different virus serotype and DENV2 were thought to be the three main factors to cause the DHF/DSS.In recent several years, various strategies for DENV vaccine development were being pursued, from attenuated viruses and inactivated viruses to DNA vaccine and recombinant preparations. Especially vaccine development focuses on tetravalencea vaccine to defend four serologic types. Although the live attenuated tetravalencea vaccine had been regarded as the most potential vaccine, unfortunately there is no an available and safe vaccine up to now.Based on above-mentioned situation of DENV infection and their clinic therapy, researchers think about passive immunity or therapy-monoclonal antibodies once more. As early as 1907, sera from individuals recovering from rubeola (measles) were used to prevent infection from this highly contagious virus. During the 1940s, improvements in the quality of fractionated human immunoglobulin led to the widespread use of intramuscular injections of pooled human immunoglobulin to prevent and treat viral infections. Many of these products are still on the market today. However, the disadvantages associated with polyclonal antibody products are that the vast majority of their constituent virus-specific antibodies are non-neutralizing,batch-to-batch variation and risk of pathogen transmission.With the development of monoclonal antibody (mAb) preparation since 1982, using specific mAb with neutralizing ability to protect and therapy virus diseases became new focus. These successes, in both industrial and academic laboratories, coupled with ongoing changes in the biomedical and regulatory environments, herald an era when the commercial development of human antiviral mAb therapies will likely surge. Thus it is a basic of the stratagem to obtain neutralizing antibodies, especially neutralizing mAbs.In the present study, purified DENV serotype 2 (DENV2) obtained from density gradient centrifugation was used as antigen to immunize BALB/c mice. After cells fusion and identification by indirect enzyme-linked immunosorbent assay (ELISA), Western-blot and indirect immunofluorescence assay (IFA), ten mAbs (78#,1E7,2B10,6C1,6H3,7F7,8B6,8F12,8H1 and 8H4) were generated and they showed different neutralizing activity to DENV2 in vitro. 78# and 8F12 had neutralizing ability and could protect suckling mice from the virus challenge partly. Our results provided insight into potential application of these mAbs for prophylactic and therapeutic purposes. The main results were as follows:RESULTS1. Preparation and verification of purified virusDENV2 (strain Tr1751) was propagated in C6/36 cells and the supernatant was obtained and stored at–80°C, and the titre of 3 litre liquid was about 1×107 PFU/ml. After PEG-8000 concentrated, the titre recovered around 2×107 PFU/ml. The suspension was loaded onto pre-formed gradients made up of 30 %, 40 %, 50 %, 55 % and 60 % (w/v) sucrose, and ultra-centrifuged at 110 000×g. At the end of sucrose density gradient centrifugation, the band between 55 %～60 % (w/v) was the main band which DENV sedimentation. The purified virus was collected for the studies behind after confirmed by SDS-PAGE and plaque assay (about 1×106 PFU/ml).2. Obtaining ten hybridoma cells which secrete the specific mAb against DENV2BALB/c mice were immunized with purified DENV2 and an equal volume of complete Freund's adjuvant. After booster immunizations and last immunization, the spleen cells from immunized mouse were fused with SP2/0 myeloma cells by PEG-4000. Then the hybridomas were selected by HAT culture medium. The supernatants of living cells were subjected to test antibody titer by indirect ELISA. Positive hybridomas were selected and cloned 5～8 times, then ten hybridomas named as 78#,1E7,2B10,6C1,6H3,7F7,8B6,8F12,8H1 and 8H4 were obtained. MAbs were harvested from hybridomas grown in 1640 medium without fetal bovine serum. The hybridomas were intraperitoneally injected to BALB/c mice for production ascites of mAbs respectively. The ascites were collected for the following examinations.3. Identification of mAbs specificity(1) Indirect ELISAThe 96-well plates were coated with purified DENV2 or bovine serum albumin. The supernatants of hybridomas were added as primary antibody respectively. Mouse anti-DENV2 sera and normal mouse sera (NMS) were used as positive and negative controls. HRP-conjugated goat anti-mouse IgG was used as secondary antibody, and coloration with o-phenylenediamine. The absorbencies were determined at 492nm. The absorbencies values of ten hybridomas were 2.1-fold higher than that in controls.(2) Saturation curve of mAbThe protocol of this experiment is same as indirct ELISA. After optimization concentration of antigen was confirmed, mAb ascites were diluted into 1:10,1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560,1:5120,1:10240,1:20480 as primary antibodies respectively. Then the saturation curve of mAbs OD492nm were drew, and from the curve we got the mAbs'degrees of saturation. We selected mAbs which recognized the same virus protein and belonged to the same subclass of Ig to undergo build-up assay. On the base of mAbs'degrees of saturation, 8F12 + 1E7 and 78# + 2B10 were chosen to build up. The results showed OD492nm step up after mAbs overlay and suggested that they recognized different epitopes.(3) Indirect immunofluorescence assay (IFA)Three-week-old female BALB/c mice were inoculated intracerebrally with DENV2. On the day of paralysis onset, the brains were taken out from the mice and then subjected to prepare freezen sections. The sections were incubated with mAb ascites as primary antibodies and then incubated with FITC-conjugated goat anti-mouse IgG as secondary antibody. The images were observed under fluorescence microscope. Anti-DENV2 sera and NMS were used as positive and negative controls respectively. Eight mAbs (78#,1E7,2B10,6C1,7F7,8B6,8F12,8H4) showed positive reaction with brain sections of DENV2-infected mice. Strong fluorescence intensity was observed in the cytoplasm of the cells in hippocampus, and the distribution of specific reaction was similar with that seen in section stained with mouse anti-DENV2 sera. However, the intensity of fluorescence of mAb was weak as compared with positive control. Specific reaction was rarely observed in section stained with two mAbs (6H3,8H1) and NMS.(4) SDS-PAGE and Western-blotThe purified virus was denatured and applied onto the gels. After electrophoresis, the gels were stained with coomassie brilliant blue or transferred onto nitrocellulose membrane for western-blot analysis. In SDS-PAGE profile, main DENV2 proteins were detected with estimated molecular masses ranging from 40 to 100 Kd. Western-blot was carried out to identity the specificity of mAbs to DENV2 proteins. Six mAbs (78#, 1E7, 2B10, 8B6, 8F12 and 8H1) reacted specifically with DENV2 E protein which relative molecular mass was about 60 Kd. One mAb (6C1) reacted specifically with DENV2 NS3 protein which relative molecular mass was about 70 Kd. Two mAbs (8H1, 6H3) reacted specifically with DENV2 NS5 protein which relative molecular mass was about 100 Kd. MAb 7F7 had no reaction with DENV2 polypeptide. In order to confirm it, the same experiment was performed using lysates of Vero cells and DENV2 infected Vero cells. As expected, the similar results were obtained.(5) Ten hybridomas were isotyped by Mouse mAb Isotyping Kit (Sigma, USA). 8F12, 1E7 and 6H3 were of subclass IgG2a; 78#, 6C1 and 2B10 were of subclass IgG2b; 8H1 and 8B6 were of subclass IgG3; 7F7 and 8H4 were of subclass IgG1. Giemsa`s staining confirmed the karyotypes of hybridomas (about 100 chromosomes).4. Plaque reduction neutralization test (PRNT)2 mg of mAb ascites (or NMS as control) were serially diluted into 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320 in Minimum Essential Medium (MEM). After heat inactivation at 56°C for 30 min, all dilutions were incubated with an equal volume of DENV2 solution containing about 100 PFU/200μl at 37°C for 1 h. Then samples (DENV2-mAb or DENV2-NMS, 200μl/well) were assayed on Vero cell monolayer under overlay medium containing 5 % FBS and 1.2 % methylcellulose. After 7 days of incubation at 37°C with 5 % CO2, cells were stained with 0.5 % crystal violet and the numbers of plaque were counted. Neutralizing titers of mAbs were expressed as the reciprocal of the maximum dilution of mAb that yielded >50 % plaque reduction (PRNT50) in the virus inoculum.According to results of PRNT, 8F12 and 78# showed neutralizing ability with PRNT50 titer 40 and 60 respectively. 8H4, 7F7, 1E7, 8B6 and 2B10 had neutralizing ability with PRNT50 titer 15, 20, 30, 30 and 35 respectively. However, 8H1, 6C1 and 6H3 had rarely neutralizing effect. When 8F12 and 78# were mixed with equal volume, the combination had strong neutralizing effect about PRNT50 titer 80 at the same experimental condition. In contrast, DENV2-NMS groups had only a little non-specific interference to DENV2 infection.5. Protection assay in suckling mice78# and 8F12 mAbs were chosen for the protection assay. Firstly, DENV2 was incubated with each mAb (or NMS, or combination of two mAbs) for 1 h at 37°C water-bath. NMS was used as negative control. Then each newborn BALB/c mouse (on the second day after delivery) was intracerebrally inoculated with 20μl mixture of DENV2-mAb or DENV2-NMS containing 500 or 104 PFU of DENV2 and 80μg mAb or NMS. And the morbidity and mortality were recorded daily. 169 suckling mice were divided into five main groups. Group A and C, and group B and D were used for the protection assay of 8F12 or 78# in either 500 or 104 PFU of DENV2 respectively. Group E test the protection of combination of 8F12 and 78# in 500 PFU of DENV2. Each main group was further divided into two subgroups for mAb and control (NMS) respectively. All infected mice of five groups were observed for two weeks.All mice in NMS groups showed disease at day 6 and dead at day 8. When each mouse was inoculated intracerebrally with DENV2-mAb mixture containing 104 PFU, they became paralysis, ruffled, slowing of activity and kyphoscoliosis during 6～8 days. And with progression of DENV2 infection, most of them showed severe sickness with anorexia, asthenia and dead at day 9. When each mouse was inoculated with DENV2-mAb mixture containing 500 PFU, they showed sickness gradually and dead at day 11. Although no mouse survived, the time to fall ill and dead in mAbs group mice were obviously delaied to control mice. Moreover, when mixture of 8F12 and 78# was incubated with 500 PFU of DENV2, the mice were protected markedly from the virus challenge. Not only survival time was longer than all the groups above, but also five mice can totally be protected in observed period (survival for 14 days).In conclusion, ten mAbs were obtained from this study and they recognize natural and denatural DENV proteins. The mAbs showed different neutralizing ability and could protect newborn mice from DENV2 challenge partly. The results demonstrated that our mAbs might be useful tools for studying the mechanism of DENV infection and basic materials for investigating therapy mAb to diseases caused by DENV.
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